About ISAE : Ethical Guidelines

Ethical Treatment of Animals in Applied Animal Behaviour Research

Applied animal behaviour studies are of fundamental importance to developing our understanding of animals. The use of animals in such studies does, however, raise important ethical issues. Many applied behaviour studies are non-invasive and require simply observing animals in the environment that they would normally be found. But, other studies might require manipulation of the animals and/or their environments. To help ISAE members make what are sometimes difficult decisions about the procedures involved in their studies, ISAE Council decided to form a Sub-Committee to write a set of ethical guidelines. The guidelines are written to be used by researchers, conference organisers, scientific reviewers, and the Ethics Committee to assess the acceptability of papers submitted for presentation at its Congresses. These guidelines should supplement the legal requirements of the location in which the research was performed. They should not be considered as an imposition on the scientific freedom of individual researchers, but rather as helping to provide an ethical framework that ISAE members may use in making decisions about the ethical acceptability of their research.

The members of the Ethics committee (as of 2016):

Anna Olsson (Chair), Portugal
Francesco De Giorgio, The Netherlands
Francois Martin, USA
Cecilie Mejdell, Norway
Franck Peron, France
Elize van Vollenhoven, South Africa
Alexandra Whittaker, Australia

The Guidelines are printed below. Any comments can be emailed to Dr. Anna Olsson (). A similar version, written for applied animal behaviour researchers in general, is published at:

Sherwin, C.M., Christiansen, S.B., Duncan, I.J.H., Erhard, H.W., Lay, D.C., Mench, J.A., O’Connor, C.E. and Petherick, C.J., 2003. Guidelines for the ethical use of animals in applied animal behaviour research. Applied Animal Behaviour Science, 81: 291-305.

Guidelines for Ethical Treatment of Animals in Applied Animal Behaviour and Welfare Research

Prepared by ISAE Ethics Committee (2002)

1. Background

The International Society for Applied Ethology (ISAE) is a professional organisation of members with primary research interests in applied studies of animal behaviour and related disciplines. Because the work of its members requires the use of animals, the Society has had a continuing interest in promoting the welfare and ethical treatment of animals being used in research. This is particularly relevant as many ISAE members are involved directly in studying animal welfare and ethics, or are involved in relevant committee work or legislative procedure. Therefore, it is believed ISAE should have a set of written guidelines to provide its members with a basis for structured self-evaluation of the ethical nature of their work, and to serve as inspiration when planning research involving the use of animals. It is the intent of the ISAE Council that these guidelines will serve to encourage appropriate research standards and may be used to assist referees in assessing the ethical nature of abstracts submitted to ISAE congresses.

ISAE members originate from many nations with very different cultures and belief systems. In addition, they conduct a wide variety of studies using disparate species in very different contexts. These guidelines have been written with these great diversities in mind and, as a consequence, are broad rather than specific. They have been written to increase awareness, encourage individual thought and stimulate discussion of the ethical issues surrounding applied animal behaviour research. The focus is therefore on the ethical principles and how to handle these, rather than a list of do’s and don’ts applicable to all scenarios.

2. Introduction

The use of animals in research and education has attracted ethical concern for many years, most notably in toxicology and bio-medical studies. More recently, ethical concerns have been raised over less invasive studies such as animal behaviour research (Mench, 2000). This raises a strong need for justification of the use of animals in behavioural research, and some guarantee that the research is conducted in an ethically acceptable manner (Driscoll and Bateson, 1988). Concerns about the use of animals in research are being voiced by both the scientific and lay communities, evident for instance by journals imposing ethical reviews of manuscripts, requirements for funding proposals to have statements that ethical guidelines will be adhered to, and the increasing public requirement for 'transparency' of research. To address these concerns before conducting behavioural research, the investigator should first assess whether the purpose of the experiment justifies the use of animals. If the purpose is found to be justified, the investigator should next consider what criteria must be met for the experiment to be acceptable. This includes an assessment of the likely pain, distress and suffering that might be caused to the animals, and an evaluation of what level of suffering can be considered acceptable in that particular context. The investigator should be able to explain and justify his/her conclusions in order to demonstrate awareness of the ethical issues and facilitate dialogue between interested parties. Finally, a critical assessment of the experimental design will promote better quality research.

3. Legislation

It is recognised that many countries already have legislation regarding the use of animals in research. These guidelines are not intended to replace or subvert this legislation. Many ISAE members are directly involved in research related to animal welfare and legislation, therefore this society is in an enviable position to be worldleaders in the implementation of codes of good conduct relating to ethical research involving animals. It is hoped these guidelines will therefore serve to promote and progress animal welfare and ethics, rather than just following 'rules' of legislation which may be minimum standards. To ensure the highest welfare and ethical standards, investigators should remain appraised of current relevant literature and conduct their research according to the spirit and letter of their local legislation as well as the spirit of the ISAE Guidelines.

4. Ethical Stand-Point & Decision Models

By definition, ISAE members conduct research on animals or have a vested interest in behavioural research. These guidelines are therefore written with an acceptance that animals can be ‘used’ for the betterment of human or non-human animal species. There are different ethical stand-points whereby the use of animals in research can be evaluated, and several models outlining the decision process relating to the ethical use of animals in research (e.g. Bateson 1986; Porter 1992; De Cock Buning and Theune, 1994). From a utilitarian stand-point, performing research involving the use of animals may be justified if certain criteria are met, such as -

Using animals for scientific purposes is only acceptable when the harm (physical or psychological) done to animals is outweighed by the benefits of the research

To determine whether the benefits of research outweigh the costs, a cost:benefit analysis can be performed. The ‘costs’ are assessed in terms of the harm likely to be experienced by the animals used in the research, and the ‘benefits’ in terms of the gains to humans, other animal species or the environment. However, the principle also implies that the ethically acceptable option is the one that provides most benefits and involves the least costs. Therefore, when planning a study, the aim should not be to simply reduce costs to a level lower than the benefits, rather, the costs should be decreased to as low as possible (see 'Reducing the Costs') and the benefits maximised as far as possible (see 'Increasing the Benefits'). We emphasise here that this cost:benefit analysis should include any distress or harm caused by housing (or other experimenter influence) prior to and subsequent to the experimental phase of the research.

Applying decision models to ones own research can be an enlightening exercise as it can help analyse the cost:benefit of research, perhaps with a fresh, external perspective. It is worth considering discussing the scientific significance and ethical issues of proposed research with colleagues in different disciplines, or lay-persons; if these people cannot be convinced a study is worth undertaking, the investigator should look carefully at the reasons they believe it should. Finally, any animal investigator should never forget to ask the absolute question - "Can I justify the use of animals in this research?”

5. Reducing the Costs

A widely accepted method for reducing the costs associated with animal research is implementation of the Three Rs, i.e. Replacement, Reduction and Refinement (Russell and Burch, 1959).

5.1 Replacement

Replacement means either that more sentient species should be replaced by less sentient ones (but see the section on Refinement below for caveats about assessing sentience), or that animals should not be used at all if the same research or related training/education can be achieved in other ways. This may be difficult to achieve in animal behaviour studies, although model animals, video-recordings, etc. can be used in some circumstances. It has been suggested (Christiansen and Sandoe, 2000) that 'replacement' in some cases can be achieved by using animals on farms, commercial establishments, during transport or in the field, rather than animals obtained specifically for the research. That a practice is standard in one context, for example, the use of certain types of housing, restraint, or management on commercial farms, does not necessarily mean that it is ethically justifiable to replicate in the laboratory if this research can be conducted in situ.

5.2 Reduction

Reduction means keeping the number of animals to the minimum necessary to achieve the aims of the research, however, the investigator should not reduce the number to so few that the results become statistically invalid. There is a tendency in research to use fewer animals if the species is exotic or expensive to maintain, but if fewer animals can be used for economic reasons without compromising scientific validity, smaller numbers can also be used on ethical grounds. The number of animals used can be minimised by several means.

5.2.1 Previous work:

If similar work has been conducted previously, this can sometimes be used to estimate the number of animals needed to produce a definite result, or the data may sometimes be included in meta-analyses. The applicability and validity of previously published research must be considered if it is to be used in this way.

5.2.2. Statistical methods:

The number of animals used can be reduced by good experimental design, appropriate observations and statistical procedures that enable several factors to be analysed with the smallest number of animals (Hunt, 1980; Still, 1982; McConway, 1992; Chiarotti and Puopolo, 2000). This will be dependent to some extent on the behaviour being studied; if a highly variable behaviour is being recorded, this will require a larger number of animals to reduce the variance to an acceptable level. Tests for statistical power can be employed to predict the smallest number of animals that need to be used in a study in cases where variance is known or can be predicted.

5.2.3 Epidemiological approach:

Sometimes it will be possible to study the spontaneous occurrence of behaviours as they occur on farms, zoos, in the wild, etc. This means no extra animals will be required for the purposes of research. Such studies can help identify which parameters are most likely involved in a particular behaviour, and can therefore help reduce the number of animals used in any further experimental study.

5.3 Refinement

The object of refinement is to reduce to an absolute minimum, the pain, distress or suffering imposed on every individual animal used. The word 'animal' (see Appendix A) is generally taken to mean higher-order animals usually thought capable of feeling pain or experiencing suffering in other ways. It should be noted that Appendix A does not differentiate between the listed species in their potential for suffering. Therefore, it is unacceptable to substitute one species for another on the list, e.g. frogs for rats, unless there is good knowledge that the former has a lower capacity for suffering and this will not invalidate the aims of the research.

5.3.1 Choice of species:

In choosing the species for a study there are several ‘-isms’ to avoid. Sizeism should be avoided; there is little evidence that smaller animals (at least within the vertebrates) are any less capable of suffering. Speciesism (between non-human animals) should also be avoided; this can occur due to the animals’ physical appearance or ecological niche of its wild counterparts. Thus, some animals might be incorrectly regarded as less capable of experiencing suffering because we find their appearance or behaviour unattractive, or because in the wild they are a pest species or live in an environment we consider undesirable, e.g. toads, rats, squid. In addition, human anthropocentricity means it is often very difficult for us to empathise with the sensory perceptions of different species (e.g. the visual perturbations caused by placing animals with ultraviolet sensitivity into environments without ultraviolet light), or the suffering of another species (e.g. the frustration of a hen about to lay an egg but unable to find a suitable nest-site).
The species chosen for a research programme should be the most appropriate for the information that the investigator wishes to gain. The choice will usually require knowledge of the problem to be investigated, the species' natural history and the animals' previous experience. Applied behaviour studies often aim to understand the responses of a particular species in a particular environment. If an inappropriate species is used, the research might therefore be invalid requiring it to be repeated using a more appropriate species, and thus making the initial study less ethically acceptable.

5.3.2 Pain, suffering and distress:

For any research programme that might involve pain, suffering or distress, the investigator should assess thoroughly whether the information gained can be justified and if non-animal alternatives (e.g. models, video-playback) might be used. Pain, suffering or distress should be minimised both in duration and magnitude to the greatest possible extent, but without jeopardising the aims of the experiment. Some species are less responsive to painful or stressful stimuli, however, this should not necessarily be taken as indicating that these species are more tolerant or do not experience pain and suffering. Animals might have evolved responses to avoid showing evidence of pain or injury, presumably to avoid being targeted by predators.

In research involving surgery, pre- and postoperative care must be implemented to reduce adverse effects both before and after the operation. Any procedure likely to cause pain should only be performed after adequate anaesthesia and with appropriate analgesia, unless either of these endangers the experimental aims. The use of neuromuscular blocking agents alone is generally unacceptable.

It should be considered that all higher-order animals (see Appendix A) have the capacity to experience pain and are capable of experiencing suffering of one kind or another. This will depend on many factors such as the species, age, sex, reproductive condition, social status, individual experience, perceptions, motivations and natural behaviour of the animal. The possibility that invertebrates such as spiders might experience pain or an analogous sensation (reviewed by Sherwin, 2001) should also be considered.

5.3.3. Housing:

Standard housing of animals in farms, zoos and laboratories is often minimalist and designed primarily for the convenience of humans. This can often result in the animals exhibiting behavioural or physiological responses indicative of reduced welfare, although it is difficult to assess how great this impact is. Several publications have ranked the severity of procedures conducted on animals in research (e.g. Morton and Griffiths, 1985; Bateson, 1991) and some include a category listing procedures/studies which it is believed cause little or no suffering. However, housing animals under minimalist standard conditions is itself likely to cause a degree of suffering - even before any experimental procedures have been conducted. It might be argued therefore, that any study which requires housing animals under standard conditions causes suffering, even if the experimental procedure itself appears to cause none. This means that in all circumstances, investigators should be able to ethically justify why an animal is being housed and/or why it is housed under particular circumstances, even if the research does not involve a procedure that causes overt pain or distress. To provide suitable housing and husbandry, investigators should consider both the quantity and quality of space they provide for their animals, and remain appraised of current relevant literature. Again, we emphasise that welfare implications of housing and husbandry conditions prior and subsequent to the experimental phase should be considered in the ethical justification of a study.

5.3.4 Identification of animals:

It is often necessary to individually identify animals. There are many methods of achieving this. Wherever possible, non-invasive methods should be used, although these tend to be more short-term and might require repeated re-application thus potentially causing further distress to animals. Invasive methods that cause minimal pain and distress (e.g. ear-tags, wing-tags) are acceptable if they are in accordance with the aims of the study. The size of the identification device or marking method relative to the body-size of the animal should be considered, and the effects this might have on behaviour or possible suffering during and subsequent to attachment/implantation. Mutilatory forms of identification (e.g. toe-amputation), or those which injure substantial amounts of tissue should be assumed to cause substantial acute and perhaps chronic pain, and would therefore generally be considered unacceptable.

5.3.5 Other “standard practices” used in husbandry:

A variety of practices that are likely to cause pain, distress or suffering are conducted routinely upon animals on farms, in laboratories, or other commercial establishments, e.g. beak-trimming, castration, chronic food deprivation, social isolation, etc. The fact that these practices are performed routinely elsewhere, does not mean they should be placed above ethical scrutiny if they are performed on animals in a research study. Indeed, many of these practices can be considered unnecessary for animals in research, so long as their omission does not contravene the validity of the study or its aims.

5.3.6. Presence of experimenters and handling:

The presence of humans can have a considerable effect on the behaviour of animals. This presence may or may not cause distress to the animals, but in either case, if this interferes with the aims of the study it reduces the validity of the research and therefore lowers the ethical acceptability. Investigators should consider the use of remote monitoring (e.g. video), or habituating the animals to the presence of humans. It should also be remembered that 'blind' studies in which the observer has no knowledge of which treatment the animal has been subjected to, reduces the likely influence of the observer and increases the validity of the research. The way in which animals are handled can have a substantial effect on their behaviour and welfare in both the short and long term. Poor handling can cause acute responses and learned aversion to handling in the future; potentially, this can invalidate the research. Investigators should familiarise themselves with the appropriate handling methods for the animals to be used.

5.3.7 Duration of the study:

In applied behaviour studies, the end-point of a study is often relatively easy to decide. For example, a study on the behavioural responses of laying hens to a novel housing system in the U.K. might extend for 62 weeks because this is the average duration that hens are housed on farms (in the UK). Data beyond 62 weeks will probably be irrelevant and therefore less ethically acceptable. On the other hand, if the work were conducted in a country where hens were routinely housed for a longer period, it would be less ethically acceptable to terminate the study before this period.

5.3.8 Final disposal and euthanasia:

At the end of a study, investigators should consider alternatives to immediate euthanasia of the animals. There are sometimes good reasons for using animals in other studies (e.g. the animals are used to being handled, familiar with the environment or procedure), but care must be taken to ensure the animals are not used repeatedly in stressful or painful experiments. Livestock might be placed onto farms, but the investigator should consider the likely responses of the animals to the change of social and physical environment, and the legal, ownership and hygiene consequences. Similarly, some species might be placed into private homes or sanctuaries.

Field-caught animals may be placed in zoos or reserves to reduce the need for further capture of wild animals, but again, the investigator should consider the likely responses of the animals to the change of social and physical environment, and the legal, ownership and hygiene consequences. Alternatively, field-caught animals may be returned to the place of capture if their ability to survive has not been impaired and release does not constitute a health or ecological hazard to existing populations.

The manner in which animals are euthanased is a significant component of the ethical acceptability of a research programme. In applied ethology studies, the method of the animals’ death will often not be under the control of the investigator, e.g. animals on farms will usually remain on the farm and will be slaughtered commercially. If the investigator has control over the method of euthanasia, factors to be considered are the likely duration of pain and distress caused by the method, and any handling the method requires. There is evidence that some methods of killing are less appropriate than others, despite their common use and approval by many legislative agencies. Investigators should read Appendix B in this regard. It should be remembered that methods of killing might be approved by legislation because of practicality and economic issues, rather than animal welfare. Death of the animal should be confirmed before the body is discarded.

5.3.9 Procedures:

End-Point of a Procedure: Deciding on the end-point of a procedure, especially when this involves obvious pain, distress or suffering, is critical for the welfare of the animal and thus the ethical justification. Investigators should consider choosing flexible end-points, e.g. in studies of aggressive or agonistic encounters, behavioural indicators of an animal accepting defeat are likely to cause less distress than encounters which have a fixed duration of interaction arbitrarily decided upon. Death is considered to be an unacceptable end-point.

Aversive Stimuli: Animals are sometimes deliberately exposed to aversive stimuli (e.g. electric shock, fear-inducing stimuli, predator-prey interactions, intra-specific competition, infanticide). If this is essential, it should be minimised in both severity and duration in accordance with achieving the aims of the experiment. The animals' perceptual and behavioural characteristics, age, experience, etc. should be considered in planning the study. Investigators should monitor such studies frequently, or preferably, constantly. At a pre-determined point, intervention should occur; the animal should be removed from the study and given appropriate treatment or euthanasia. Barriers or escape routes should be provided for the animal to avoid the aversive stimulus where this is in accordance with achieving the aims of the experiment. Investigators should be aware of indicators of extreme fear, e.g. learned helplessness, and that some species may sometimes become totally unresponsive although aware and cognisant of their surroundings (tonic immobility). Field studies should be considered as an alternative method of investigation.

Deprivation: Animals are sometimes deprived of various resources for a variety of reasons. These resources can be of various types, e.g. social contact, straw, perches, food, water, comfort behaviours, suitable light. If deprivation is essential, this should be minimised in both severity and duration in accordance with achieving the aims of the experiment. Food is sometimes withdrawn to motivate animals to perform a particular task, however, the use of highly attractive foods or other rewards is often a more acceptable alternative (there is also evidence that food deprivation can interfere with some learning tasks (e.g. Nicol and Pope 1993; Sherwin et al., 2001)). In general, to avoid chronic hunger, it is preferable to deprive an animal of food for a pre-determined period of time before testing, rather than attempting to achieve and maintain an arbitrarily specified target bodyweight.

Adverse Conditions: Studies aimed at inducing adverse conditions in animals are sometimes conducted to gain knowledge of applied problems, e.g. parasite loads, pesticides or homeostatic challenges. These procedures may cause suffering and again should be minimised in both severity and duration in accordance with achieving the aims of the experiment. Investigators should plan frequent or constant monitoring of such studies, and appropriate intervention at a pre-determined end-point with appropriate care or euthanasia of the animals. Investigators should also consider experimental designs that allow removal of the adverse condition rather than its addition (e.g. the use of a novel insecticide on a population of sheep for which it is known that ecto-parasite burdens are already high), or naturally occurring instances of the adverse conditions.

Isolation and Crowding: Many applied behaviour studies investigate the effects of isolation or crowded conditions that are used routinely on farms and in laboratories. It should be realised that although such housing might be considered standard in some contexts, these systems may be extremely stressful to animals (see 'Housing' above). The degree of stress experienced will be markedly influenced by the species, age, sex, reproductive condition, social status, individual experience and natural behaviour of the animal. These factors should all be considered to minimise the stress likely to be experienced by the animal.

6. Increasing the Benefits

As stated previously, the benefits of any proposed research should be made as great as possible. These can be maximised in several ways.

6.1 Achieving the aims

The aims of the research should be achievable. This can be ensured by closely examining the aims and determining if the appropriate animals, equipment, housing and trained personnel are all of an adequate standard and available for the duration of the study.

6.2 Significance of the aims

The aims can be of various forms, for instance, health or welfare of humans or non-human animals, economic gains for livestock, conservation, pest control or fundamental knowledge. In applied behaviour studies it is often possible to quantify and state the likely benefits resulting from research, and therefore the significance of the aims. For example, a study on feather-pecking might be able to state the average incidence of hens pecked, the average number of injuries, mortality rates and the cost of increased food consumption as the animals attempt to maintain their body temperature. Such information helps indicate the severity and extent of the problem being addressed, and therefore the likely significance of the findings. It may be more difficult to state the likely benefits resulting from fundamental research. However, although it may be hard to predict what the potential gain of the knowledge could be, fundamental research may provide essential information and possibly even support progress in the applied field.

6.3 Researching previous work

Investigators should thoroughly familiarise themselves with previously published relevant literature. This avoids unnecessarily duplicating research (assuming previous work was done correctly), although duplication may be required in pilot studies of a novel method. It will also be possible to gauge the variability of responses and ensure the experimental design is optimised to achieve the aims of the study by using the least number of animals.

6.4 Reporting of the Study

A fundamental component of the ethical justification of animal behaviour research is the communication of results. The investigator has an ethical obligation to attempt to publish the results as completely, widely and as accurately as possible. Doing this decreases the probability of more animals being used in unnecessary duplicate studies to generate similar, redundant data. Widespread (global) communication of results is a ‘benefit’ factor in many models of the ethical assessment of animals and thus communication of results increases the benefits of the work. To demonstrate that an ethical assessment has been made, the ethical justification for choice of research and experimental design can be included. This will promote understanding and communication concerning the ethical issues and dilemmas in research involving animals (Christiansen and Sandoe, 2000).

7. Field Experiments

Investigators conducting field experiments of applied animal behaviour should consider the ethical issues discussed above, and in addition, the impact of their work on other populations of animals and ecosystems. Methods of marking, the taking of physiological samples, capture, continuous observation, etc, might all influence an animal's ability to survive both at the time of observation and in the future. The welfare of other animals dependent on the subject (e.g. offspring) should also be considered. Cuthill (1991) and Kirkwood and Sainsbury (1996) discuss ethical issues of conducting field experiments.

8. References

Bateson, P. (1986). When to experiment on animals. New Scientist, 1496: 30-32

Bateson, P. (1991). Assessment of pain in animals. Animal Behaviour, 42: 827-839

Chiarotti, F. and Puopolo, M. (2000). Refinement in behavioural research: a statistical approach. In: Progress in Reduction, Refinement and Replacement of Animal Experimentation. Eds. M. Balls, A.-M. van Zeller, and M. Halder. Elsevier, The Netherlands, pp. 1222-1238.

Christiansen, S.B. and Sandoe, P. (2000). Ethics in animal behaviour and welfare research. Proc. 34th Int Congress ISAE, Florianopolis, Brazil. Oct 17-20. p. 88

Cuthill, I. (1991). Field experiments in animal behaviour: methods and ethics. Animal Behaviour 42: 1007-1014
De Cock Buning, T. and Theune, E. (1994). A comparison of three models for ethical evaluation of proposed animal experiments. Animal Welfare, 3: 107-128

Driscoll, J.W. and Bateson, P. (1988). Animals in behavioural research. Animal Behaviour, 36: 1569-1574
Hunt, P. (1980). Experimental choice. In: The Reduction and Prevention of Suffering in Animal Experiments, RSPCA, Horsham, UK,. pp. 63-75

Kirkwood, J.K. and Sainsbury, A.W. (1996). Ethics of interventions for the welfare of free-living wild animals. Animal Welfare, 5: 235-243

McConway, K. (1992). The number of subjects in animal behaviour experiments: is Still still right? Ethics in Research on Animal Behaviour, (eds M. Stamp Dawkins and L.M. Gosling) Academic Press, London, UK. pp. 35-38

Mench, J.A. (2000). Refinement in behavioural research. In: Progress in Reduction, Refinement and Replacement of Animal Experimentation. Eds. M. Balls, A.-M. van Zeller, and M. Halder. Elsevier, The Netherlands, pp. 1213-1221

Morton, D.M.and Griffiths, P.H.M. (1985). Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment. Veterinary Record, 116: 431-436

Nicol, C.J. and Pope, S.J (1993). Food deprivation during observation reduces social learning in hens. Animal Behaviour, 45: 193-196

Porter, D.G. (1992). Ethical scores for animal experiments. Nature, 356: 101-102

Russell, W.M.S. and Burch, R.L. (1959). The Principles of Humane Experimental Technique. Methuen & Co Ltd., London, UK.

Sherwin, C.M. (2001). Can invertebrates suffer? Or how robust is argument-by-analogy? Animal Welfare, 10 (suppl) 103-118

Sherwin, C.M., Heyes, C.M., Leeb, C. and Nicol, C.J. (2001). The effect of demonstrator reward on social learning of key pecking by domestic hens. Proc. ISAE UK Congress, York, April 10th 2001

Still, A.W. (1982). On the number of subjects used in animal behaviour experiments. Animal Behaviour, 30: 873-880

Appendix A

Higher-Order Animals

Higher-order animals include all vertebrates and, of the invertebrates, some members of the phylum Mollusca (e.g. octopus, squid) and some members of the phylum Arthropoda (crab, lobster, crayfish). Higher-order animals are also considered to include mammalian foetuses during the last half of pregnancy, un-hatched young of the species stated above during the last half of their development in the egg, and marsupial pouch young. This is because it is thought likely that animals in these stages of development might be able to experience pain and suffering.

(This classification of higher-order animals is adapted from New Zealand law which regulates the scientific use of animals, Animal Welfare Act 1999).

Appendix B

Comments on some sommonly used methods of killing

ISAE members often conduct research with farm or laboratory species. When commercially reared, these species are often killed in great numbers and methods are used which take into account factors such as cost, speed of throughput, and practicality; the welfare of the animals may be given a lower priority than would be otherwise. When these species are killed after behavioural studies, the numbers involved are usually considerably lower than after rearing on farms or in commercial laboratories, and the animals are often very accustomed to being handled by humans. This makes alternative methods of euthanasia more practical and economically justifiable - or alternatively, more time to correctly administer the method of killing. A review of the literature reveals that some legally accepted methods of killing used commercially might have associated with them welfare concerns that appear to be widely unknown or perhaps ignored (see below), making these less ethically appropriate for use on animals used in small-scale research studies. We should remember, the term 'euthanasia' generally refers to 'an easy and painless death' or 'the killing of an animal with a minimum of physical and mental suffering, depending on the species' (Close et al., 1996), suggesting legally accepted methods might not always be described as 'euthanasia'. Investigators are strongly encouraged to read Close et al., (1996) and the AVMA Panel on Euthanasia (2001).

Decapitation and cervical dislocation:

There is evidence that decapitation and cervical dislocation may not render an animal immediately unconscious. In rats, normal brain electrical activity indicative of consciousness can persist for 30s after decapitation, and visual responses can be evoked from hens for 30s after decapitation (Gregory and Wotton, 1986) (see also Anon (2000) and Holson (1992)). Reptilian brain metabolism can function at low respiration and heart rates, and Warwick (1986) cites evidence that the heads of snakes will respond to approach for up to 59 mins after decapitation. Cervical dislocation must achieve severance of the spinal cord from the brain (to prevent neural transmission) and all of the major blood vessels in the neck (to prevent blood supply to the brain). Nevertheless, cervical dislocation raises concerns about animal welfare; visual evoked responses can be obtained from hens for up to 4 minutes after dislocation, depending on the method (stretching the neck causes loss of the response more quickly than crushing, allegedly due to concussion of the brain) (Gregory and Wotton, 1990). It is therefore preferable that if an animal is to be killed by decapitation or cervical dislocation, it should be rendered unconscious prior to this.

Gaseous anaesthetic overdose:

Exposure to carbon dioxide is a method of killing that is widely recommended for small farm and laboratory animals and birds, however, there are aspects of its physical characteristics and the physiological responses induced by this gas that raise welfare concerns. CO2 is an acidic gas that is known to cause irritation to mucus membranes, it has a pungent odour and can cause a profound sense of breathlessness before inducing unconsciousness in humans. Inhalation of this gas can cause animals to exhibit an excitation phase - this might be caused by the animal possibly experiencing a sensation analogous to suffocation (respiratory systems are stimulated by increasing CO2 concentrations in the blood) - although there is debate whether the animals might already have become unconscious at this point (Jongman et al., 2000). Pigs will voluntarily experience 72 hrs water deprivation or 24 hrs food deprivation rather than experience a second exposure to CO2 (Raj and Gregory, 1995; see also Jongman et al., 2000). The degree of aversion to CO2 varies with concentration (Raj and Gregory, 1995; see also Hewett et al., 1993; Hackbarth et al., 2000) and aversion has also been reported in several other species, e.g. aquatic mammals and birds (Raj, 1996; Cooper et al., 1998), thus CO2 aversion is both species- and concentration dependent.

Hypoxia or anoxia:

Various gases have been used to deplete oxygen and so cause hypoxia or anoxia. Evidence suggests that hypoxia or anoxia is a humane way of inducing unconsciousness and death, but problems may arise when the gas used as a substitute has aversive properties or the species of animals to be euthanased has physiological compensatory mechanisms (resilience or tolerance to hypoxia).

Argon is odourless, tasteless and appears to cause no aversion in pigs. A mixture of 30% CO2 in argon causes loss of brain function in chickens, turkeys and pigs (Raj, Wotton and Gregory, 1992; Raj & Gregory, 1994; Raj et al., 1997).

Nitrogen has also been used to induce anoxia. Because this has a density very similar to air it must be used in a closed container. A mixture of mostly nitrogen (>80 % by volume) and a small proportion of argon has been recently implemented for killing broilers under commercial conditions (Raj, A. B. M. pers. comm.).

Carbon monoxide causes rapid death through hypoxia and there is little apparent distress caused during the induction of unconsciousness in terrestrial species. CO must be supplied using a commercial source of 100% because other sources usually contain additional substances that might interfere with the effectiveness of CO hypoxia. CO is toxic to humans and therefore the concentration both inside and outside the chamber must be monitored.

Hypoxia (induced by CO, nitrogen or argon) can cause vocalisations and convulsions after loss of consciousness that may be aesthetically unpleasant. Due to physiological and metabolic differences, hypoxia may not be the best killing method for rodents, reptiles, aquatic animals and birds.


Close et al., (1996) and the AVMA Panel on Euthanasia (2001) list a wide variety of methods considered to be unacceptable as methods of killing vertebrates. These included hypothermia (e.g. ice-slurries for fish and other heterotherms), hyperthermia, drowning or removal from water, neck crushing, nitrous oxide, cyclopropane, ether, chloroform, sedatives (due to the large volume required), certain orally administered agents and narcotic analgesics.

References & Further Reading

American Veterinary Medical Association (AVMA) Panel on Euthanasia (2001). Journal of the American Veterinary Medical Association 218: 669-696.

Anon. (2000) New Scientist, Dec 16th, p. 101

Close, B., Banister, K., Baumans., V., Bernoth, E.M., Bromage, N., Bunyan, J., Erhardt, W., Flecknell, P., Gregory, N., Hackbarth, H., Morton, D. and Warwick, C. (1996). Recommendations for euthanasia of experimental animals. Laboratory Animals, 30: 293-316

Cooper, J., Mason, G. and Raj, M. (1998) Determination of the aversion of farmed mink (Mustela vision) to carbon dioxide. Veterinary Record, 143: 359-361

Gregory, N.G. and Wotton, S.B. (1986). Effect of slaughter on the spontaneous and evoked activity of the brain. British Poultry Science, 27: 195-205

Gregory, N.G. and Wotton, S.B. (1990). Comparison of neck dislocation and percussion of the head on visual evoked responses in the chicken's brain. Veterinary Record, 126: 570-572

Hackbarth, H., Kuppers, N. and Bohnet, W. (2000). Euthanasia of rats with carbon dioxide - animal welfare aspects. Laboratory Animals, 34: 91-96

Hewett, T.A., Kovacs, M.S., Artwohl, J.E. and Bennet, B.T. (1993). A comparison of euthanasia methods in rats using carbon-dioxide in prefilled and fixed flow-rate filled chambers. Laboratory Animal Science, 43: 579-582

Holson RR (1992). Euthanasia by decapitation - evidence that this technique produces prompt, painless unconsciousness in laboratory rodents. Neurotoxicology and Teratology 14: 253-257

Jongman, E.C., Barnett, J.L. and Hemsworth, P.H. (2000). The aversiveness of carbon dioxide stunning in pigs and a comparison of the CO2 stunner crate vs. the V-restrainer. Applied Animal Behaviour Science, 67: 67-76

Raj, A. B. M. (1996). Aversive reactions of turkeys to argon, carbon dioxide and a mixture of carbon dioxide and argon. Veterinary Record, 138: 592-593.

Raj, A.B.M. and Gregory, N.G. (1993). Time to loss of somatosensory evoked potentials and the onset of changes in the spontaneous electroencephalogram of turkeys during gas stunning. Veterinary Record, 133: 318-320

Raj, A. B. M. and Gregory, N. G. 1994. An evaluation of humane gas stunning methods for turkeys. The Veterinary Record, 135: 222-223.

Raj, A.B.M. and Gregory, N.G. (1995). Welfare implications of the gas stunning of pigs 1. Determination of aversion to the initial inhalation of carbon dioxide or argon. Animal Welfare, 4: 273-280

Raj, A. B. M., Wotton, S. B. and Gregory, N. G. 1992. Changes in the somatosensory evoked potentials and spontaneous electroencephalogram of hens during stunning with a carbon dioxide and argon mixture. British Veterinary Journal, 148: 147-156.

Raj, A. B. M., Johnson, S. P., Wotton, S. B. and McInstry, J. L. 1997. The Veterinary Journal, 153: 329-340

Reilly J.S. (editor) (1993) Euthanasia of Animals Used for Scientific Purposes. ANZCCART, Glen Osmond, South Australia
UFAW Handbook On the Care and Management of Laboratory Animals, Vols 1 and 2 (1999).


Warwick, C. (1986). Euthanasia of reptiles - decapitation - an inhumane method of slaughter for the class reptilia. Canadian Veterinary Journal, 27: 34-34

Appendix C

Further Reading & Information

Ethics/Codes of practice/Guidelines

Anon., (2001). Guidelines for psychologists working with animals. Quarterly Journal of Experimental Psychology, Section B - Comparative and Physiological Psychology, 54: 81-91

Animal Behavior Society/Association for the Study of Animal Behaviour (2001). Guidelines for the treatment of animals in behavioural research and teaching. Animal Behaviour, 61: 271-275

Dolan, K. (1999). Ethics, Animals and Science. Blackwell Science Ltd, UK.
Fraser, D. (1995). Science, values and animal welfare: Exploring the 'inextricable connection'. Animal Welfare, 4: 103-117

Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. (1999) Federation of Animal Science Societies, Savoy, IL, USA Guide for the Care and Use of Laboratory Animals (1996). Institute of Laboratory Animal Resources. National Academy Press, Washington, D.C., USA Guidelines for the Use of Animals in Neuroscience Research. XXX

Hart. L.A. (ed) (1998). Responsible Conduct with Animals in Research. Oxford University Press, New York, USA.

Monamy, V. (1996). Animal Experimentation: A Student Guide to Balancing the Issues.

Orlans, F.B., Beauchamp, T.L., Dresser, R., Morton, D.B. and Gluck, J.P. (1998). The Human Use of Animals - case studies in ethical choice. Oxford University Press, New York, USA.

Rowan, A.N. (1984). Of Mice, Models, and Men: a critical evaluation of animal research. State University of New York Press, USA.

Sandøe, P. (1994). Animal Research and Ethics. In: Handbook of Laboratory Animal Science (eds P. Svendsen and J. Hau) pp. 1-9, CRC Press LLC, Florida, USA.

Smith, J.A. and Boyd, K.M. (eds) (1991). Lives in the Balance: The Ethics of Using Animals in Biomedical Research. Oxford University Press, Oxford, UK.

Stamp Dawkins, M. and Gosling L.M.(eds) (1991). Ethics in Research on Animal Behaviour. Academic Press, London UK. (see Animal Behaviour, 41: 183-186)


Animal Numbers

Cohen, J. (1977). Statistical Power Analysis for the Behavioral Science. Academic Press, New York, USA.
Engeman, R.M.and Shumake, K. (1993). Animal welfare and the statistical consultant. American Statistician, 47: 229-233

Festing, M.F.W. (1994). Reduction of animal use: experimental design and quality of experiments. Laboratory Animals, 28: 212-221

McCance, I. (1989). The number of animals. News in Physiological Science, 4: 172-176


Pain, Suffering and Distress

Flecknell, P.A. (1986). Recognition and alleviation of pain in animals. In, Advances in Animal Welfare Science. 1985/1986 (eds M.W.Fox, and L.D. Mickley) pp. 61-77, Martinus Nijhoff, Boston, USA.
Gentle, M.J. (1992). Pain in birds. Animal Welfare, 1: 235-247

Melzack, R. and Wall, P. (1982). The Challenge of Pain. Penguin, Hardmondsworth, UK.
Rachlin, H. (1985). Pain and behavior. Behavioural Brain Sciences, 8: 43-83

Rollin, B.E. (1986). Animal pain. In, Advances in Animal Welfare Science 1985/1986 (eds M.W.Fox, and L.D. Mickley), Martinus Nijhoff, Boston, USA.

Stafleu, F.F., Rivas, E., Rivas, T. Vorstenbosch, J. Heeger, F.R. and Beynen, A.C. (1992). The use of analagous reasoning for assessing discomfort in laboratory animals. Animal Welfare, 1: 77-84

General Animal Welfare

Appleby, M.C. and Hughes, B.O. (eds) (1997). Animal Welfare. CAB International, Wallingford, UK.
Broom, D.M. and Johnson, K.G. (1993). Stress and Animal Welfare. Chapman and Hall, London, U.K.
Duncan, I.J.H., 1996. Animal welfare defined in terms of feelings. Acta Agric. Scand., Sect. A, Anim. Sci., Suppl. 27, 29-35

Fraser, D. and Duncan, I.J.H. (1998). 'Pleasures', 'pains' and animal welfare: toward a natural history of affect. Animal Welfare, 7: 383-396

Gregory, N.G. (1998). Physiological mechanisms causing sickness behaviour and suffering in diseased animals. Animal Welfare, 7: 293-305

Mason, G. and Mendl, M. (1993). Why is there no simple way of measuring animal welfare? Animal Welfare, 2: 301-319

Wiepkama, P.R. and Koolhaas, J.M. (1993). Stress and animal welfare. Animal Welfare, 2: 195-218




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